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Although CD133 has been proposed as a marker for brain tumor–initiating cells, studies show that a tumorigenic potential exists among CD133− glioma cells as well. However, it is not established whether the ability of CD133− cells to form tumors is a property confined to a small subpopulation, rather than a common trait associated with most glioma cell types. Thus, we used lentiviral vectors expressing green fluorescent protein under lineage-specific promoters to identify CD133− glioma cells expressing Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE). Flow cytometry analysis showed the presence of CD133− subpopulations expressing these markers in glioma cell lines and in primary cultures from human glioblastoma (GBM) biopsies. Moreover, analysis of cell cycle distribution showed that subgroups expressing Nestin, GFAP, and NSE uniformly contained actively cycling cells, when cultured in serum-containing medium and stem cell medium. These subpopulations were fluorescence-activated cell sorted from CD133− U373 glioma cells and implanted intracerebrally in severe combined immunodeficient mice. Moreover, we implanted Nestin-, GFAP-, and NSE-positive glioma cells sorted from a human GBM biopsy, following removal of CD133-positive cells. All the CD133− subpopulations produced tumors, with no significant differences in survival or tumor take rates. However, there was a trend toward lower take rates for CD133− glioma subpopulations expressing GFAP and NSE. These findings suggest that the ability to form tumors may be a general trait associated with different glioma cell phenotypes, rather than a property limited to an exclusive subpopulation of glioma stem cells. Cancer Res; 70(11); 4274–9. ©2010 AACR.

Supplementary Methods, Figure Legends 1-2 from Glioma Cell Populations Grouped by Different Cell Type Markers Drive Brain Tumor Growth

Supplementary Figure 1 from Glioma Cell Populations Grouped by Different Cell Type Markers Drive Brain Tumor Growth

Supplementary Figure 2 from Glioma Cell Populations Grouped by Different Cell Type Markers Drive Brain Tumor Growth

Flavokawain B (FKB), a natural kava chalcone, displays potent antitumor activity in various types of cancer. The mechanism of action, however, remains unclear. Here, we evaluated the efficacy of FKB in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its inhibitory effects in cancer. Approximately 60% of GBM cells became senescent after treatment with FKB as assessed in the senescence-associated (SA)-GLB1/SA-β-galactosidase assay. The cellular process of autophagy potentially contributed to the establishment of senescence. Transmission electron microscopy revealed the formation of autophagic vesicles under FKB treatment, and MAP1LC3B (microtubule associated protein 1 light chain 3 beta)-II was increased. Transfection of ATG5 or ATG7 small interfering RNAs (siRNAs) inhibited FKB-induced autophagy in U251 cells. Western blot revealed that molecular components of the endoplasmic reticulum stress pathway were activated, including ATF4 (activating transcription factor 4) and DDIT3 (DNA damage inducible transcript 3), while levels of TRIB3 (tribbles pseudokinase 3) increased. In addition, based on the phosphorylation status, the AKT-MTOR-RPS6KB1 pathway was inhibited, which induced autophagy in GBM cells. Inhibition of autophagy by autophagy inhibitors 3-methyladenine and chloroquine or knockdown of ATG5 or ATG7 caused FKB-treated U251 cells to switch from senescence to apoptosis. Finally, knockdown of ATG5 or treatment with chloroquine in combination with FKB, significantly inhibited tumor growth in vivo. Our results demonstrated that FKB induced protective autophagy through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in GBM cells, indicating that the combination treatment of FKB with autophagy inhibitors may potentially be an effective therapeutic strategy for GBM. Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; ATG: autophagy related; CASP3: caspase 3; CCK-8: cell counting kit-8; CDKN1A: cyclin-dependent kinase inhibitor 1A; CQ: chloroquine; DDIT3: DNA damage inducible transcript 3; DMEM: Dulbecco’s modified Eagle’s medium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FKB: flavokawain B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBM: glioblastoma multiforme; GFP: green fluorescent protein; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; 1RPS6KB1: ribosomal protein S6 kinase B1; SA-GLB1: senescence-associated galactosidase beta 1; siRNA: short interfering RNA; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TRIB3: tribbles pseudokinase 3; TUNEL: deoxynucleotidyl transferase-mediated dUTP nick-end labeling

// Jiwei Wang 1 , Qichao Qi 1 , Zichao Feng 1 , Xin Zhang 1 , Bin Huang 1 , Anjing Chen 1 , Lars Prestegarden 2, 3 , Xingang Li 1 , Jian Wang 1, 2 1 Department of Neurosurgery, Qilu Hospital of Shandong University and Brain Science Research Institute, Shandong University, Jinan, 250012, P.R. China 2 Department of Biomedicine, University of Bergen, 5009 Bergen, Norway 3 Department of Dermatology, University of Bergen, 5021 Bergen, Norway Correspondence to: Xingang Li, email: lixg@sdu.edu.cn Jian Wang, email: jian.wang@uib.no Keywords : berberine, glioblastoma, autophagy, apoptosis, metabolism Received: March 15, 2016 Accepted: August 09, 2016 Published: August 19, 2016 ABSTRACT There is an urgent need for new therapeutic strategies for patients with glioblastoma multiforme (GBM). Previous studies have shown that berberine (BBR), a natural plant alkaloid, has potent anti-tumor activity. However, the mechanisms leading to cancer cell death have not been clearly elucidated. In this study, we show that BBR has profound effects on the metabolic state of GBM cells, leading to high autophagy flux and impaired glycolytic capacity. Functionally, these alterations reduce the invasive properties, proliferative potential and induce apoptotic cell death. The molecular alterations preceding these changes are characterized by inhibition of the AMPK/mTOR/ULK1 pathway. Finally, we demonstrate that BBR significantly reduces tumor growth in vivo , demonstrating the potential clinical benefits for autophagy modulating plant alkaloids in cancer therapy.

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

Background: Resistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Autophagy inhibitors have been shown to enhance the efficacy of radiotherapy for certain solid tumors. However, current inhibitors do not penetrate the blood-brain-barrier (BBB). Here, we assessed the radiosensitivity effects of the antipsychotic drug trifluoperazine (TFP) on GBM in vitro and in vivo. Methods: U251 and U87 GBM cell lines as well as GBM cells from a primary human biopsy (P3), were used in vitro and in vivo to evaluate the efficacy of TFP treatment. Viability and cytotoxicity was evaluated by CCK-8 and clonogenic formation assays. Molecular studies using immunohistochemistry, western blots, immunofluorescence and qPCR were used to gain mechanistic insight into the biological activity of TFP. Preclinical therapeutic efficacy was evaluated in orthotopic xenograft mouse models. Results: IC50 values of U251, U87 and P3 cells treated with TFP were 16, 15 and 15.5 μM, respectively. TFP increased the expression of LC3B-II and p62, indicating a potential disruption of autophagy flux. These results were further substantiated by a decreased Lysotracker Red uptake, indicating impaired acidification of the lysosomes. We show that TFP and radiation had an additive effect when combined. This effect was in part due to impaired TFP-induced homologous recombination. Mechanistically we show that down-regulation of cathepsin L might explain the radiosensitivity effect of TFP. Finally, combining TFP and radiation resulted in a significant antitumor effect in orthotopic GBM xenograft models. Conclusions: This study provides a strong rationale for further clinical studies exploring the combination therapy of TFP and radiation to treat GBM patients. publishedVersion

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1101-1) contains supplementary material, which is available to authorized users.

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O6-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling. Electronic supplementary material The online version of this article (doi:10.1007/s00401-011-0867-2) contains supplementary material, which is available to authorized users.

Although CD133 has been proposed as a marker for brain tumor–initiating cells, studies show that a tumorigenic potential exists among CD133− glioma cells as well. However, it is not established whether the ability of CD133− cells to form tumors is a property confined to a small subpopulation, rather than a common trait associated with most glioma cell types. Thus, we used lentiviral vectors expressing green fluorescent protein under lineage-specific promoters to identify CD133− glioma cells expressing Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE). Flow cytometry analysis showed the presence of CD133− subpopulations expressing these markers in glioma cell lines and in primary cultures from human glioblastoma (GBM) biopsies. Moreover, analysis of cell cycle distribution showed that subgroups expressing Nestin, GFAP, and NSE uniformly contained actively cycling cells, when cultured in serum-containing medium and stem cell medium. These subpopulations were fluorescence-activated cell sorted from CD133− U373 glioma cells and implanted intracerebrally in severe combined immunodeficient mice. Moreover, we implanted Nestin-, GFAP-, and NSE-positive glioma cells sorted from a human GBM biopsy, following removal of CD133-positive cells. All the CD133− subpopulations produced tumors, with no significant differences in survival or tumor take rates. However, there was a trend toward lower take rates for CD133− glioma subpopulations expressing GFAP and NSE. These findings suggest that the ability to form tumors may be a general trait associated with different glioma cell phenotypes, rather than a property limited to an exclusive subpopulation of glioma stem cells. Cancer Res; 70(11); 4274–9. ©2010 AACR.

Array comparative genomic hybridization (aCGH) is a powerful tool to detect relative DNA copy number at a resolution limited only by the coverage of bacterial artificial chromosomes (BACs) used to print the genomic array. The amount of DNA needed to perform a reliable aCGH analysis has been a limiting factor, especially on minute tissue samples where limited DNA is available. Here we report a simple, highly sensitive and reliable aCGH method to analyze samples of no more than 1 ng genomic DNA. The speed and simplicity of the technique are ideal for studies on small clinical samples such as needle biopsies.

Despite major therapeutic advances in the management of patients with metastatic melanoma, brain metastases remain an intractable problem. It has proven difficult to identify and target single genes or pathways critical to brain metastasis formation. Here, we explore a more global and preventive treatment approach to melanoma brain metastases within an established xenograft model of human melanoma. Mice were injected intracardially and followed for six weeks with whole-body BLI and brain MRI. Tumor-bearing brains, adrenals, ovaries, and femurs were harvested and enzymatically dissociated before three tumor cell samples from each organ were flow-sorted and subjected to RNA sequencing. We were able to define a 108-gene brain metastasis gene signature, and queried the Connectivity Map database for candidate drugs, i.e. drugs that induce an opposite gene expression profile in various cancer cell lines. We found the cholesterol analogue beta-sitosterol to effectively inhibit brain metastases and improve survival, both in established and preventive therapeutic settings. Beta-sitosterol extensively suppressed the MAPK pathway, and effectively reduced mitochondrial respiration through Complex I inhibition. This novel mechanistic synergy may open new avenues of systemic therapy against metastatic melanoma, as increased mitochondrial respiration is a key mediator of resistance to MAPK-targeted drugs.

Tumour spheroids initiated from glioma biopsy specimens provide a valuable three-dimensional cell culture system that share several biological features of malignant brain tumours in situ. Upon xenotransplantation in immunodeficient rats, tumours derived from such spheroids exhibit a highly infiltrative growth. Successful cryopreservation of spheroid specimens therefore represents an excellent tool for future comparative studies of tumour growth and progression. Thus, if frozen stocks of human glioma spheroids can be established, similar to those obtained from cancer cell lines, it would ease the planning of biopsy-based experiments. In this context, it is crucial that cryopreservation does not alter the biological behaviour of the tumour spheroids. The biopsy spheroids were frozen to -40 degrees Celsius, stored for 1 week at -196 degrees Celsius, thawed rapidly and cultured for 1 week. The viability of the spheroids was compared against controls using a two-colour fluorescence assay, which demonstrated that cryopreservation was well tolerated. Using an in vitro invasion assay, it is shown that the freezing procedures did not affect the spheroids ability to invade a collagen gel. Cryopreserved and control tumour spheroids were equally tumourogenic, and produced overlapping survival curves when transplanted into the brains of immunocompromised rats. Immunohistochemical analyses showed no significant changes regarding microvessel density or proliferation index. Furthermore, gene expression profiling using a macroarray system detected no significant changes following cryopreservation. The present data show that cryopreservation is well tolerated, and represent a methodologically reliable storage method for biopsy spheroids that can be used in experimental studies at later time points.

Two phase III clinical trials with temozolomide (TMZ) in combination with bevacizumab for newly diagnosed glioblastoma multiforme (GBM) failed to demonstrate any survival benefit when compared to TMZ alone. Previous studies have shown that bevacizumab treatment increases intratumoral hypoxia followed by up-regulation of hypoxia-inducible factor 1 alpha (HIF-1α), a key mediator of tumor cell survival under hypoxic conditions. As such, inhibition of HIF-1α might be a promising therapeutic strategy in GBM. The aim of this study was to identify new pharmacological inhibitors of HIF-1α by combining functional genomics data with the Connectivity Map (cMap) database. First, a genome-wide lentiviral short hairpin RNA (shRNA) synthetic lethality screen was performed using a pool of 27,290 shRNAs targeting 5,046 known human genes to identify genes essential for GBM cell survival under hypoxia. The cMap database was subsequently interrogated using these results to identify drugs that would induce corresponding changes in gene expression. By applying this approach, we identified albendazole (ABZ), a widely used anti-helmintic drug, as a potent inhibitor of HIF-1α. ABZ treatment of GBM cells induced a broad-spectrum down-regulation of pro-angiogenic-factors, such as vascular endothelial growth factor (VEGF). Furthermore, ABZ induced a G2/M cell cycle arrest followed by apoptotic cell death. Mechanistically, we showed that ABZ disrupts microtubule polymerization leading to impaired translation of HIF-1α mRNA. The therapeutic potential of ABZ and concomitant bevacizumab is currently being assessed in vivo and the data will be presented. In conclusion, ABZ is a potent pharmacological inhibitor of HIF-1α. Considering that HIF-1α is a key mediator of resistance to anti-angiogenic therapy, a combined approach with ABZ and bevacizumab might be a promising treatment strategy for GBM patients.

Glioblastomas are highly resistant to therapy and carries a dismal prognosis. Standard of care includes surgery followed by radiotherapy (RT) with concurrent and adjuvant chemotherapy with temozolomide (TMZ). The addition of TMZ to RT increases the overall survival by approximately 3 months compared to RT alone. In this work we sought to identify synthetic lethal interactions that could potentiate TMZ treatment. By integrating results from a genome-wide lentiviral shRNA library screen with the Connectivity Map database (Broad Institute), we have identified signaling networks critical for cancer cell survival during therapy. We transfected the human GBM cell lines with a pooled RNAi library targeting approximately 5000 genes, and incubated the cells at three sub-lethal TMZ concentrations or in control medium. Individual silencing of 292 genes lead to significantly reduced cell growth in the treated population while silencing of 414 genes increased cellular viability compared to the control group. The Connectivity Map database was then interrogated using these results to identify small-molecule inhibitors that would induce analogous changes in gene expression. From this approach we identified Thioridazine, a dopamine 2-receptor (DRD2) antagonist, as a potent sensitizer for TMZ, reducing the LC50 about 20-fold. shRNA mediated downregulation of DRD2 validated these findings. Moreover, we show mechanistically that the therapeutic action of Thioridazine was mediated by autophagic cell death. Genetic and pharmacological interrogation of downstream cell signaling events identifed an activation of the DRD2/β-arrestin/Akt/mTor pathway. The therapeutic potential of Thioridazine and concomitant TMZ in vivo will be described. We believe that DRD2 represent a nodal point connecting autocrine survival cues in a subset of glioblastomas. Our findings suggest that, like androgen and estrogen dependence in prostate- and breast cancer, dopamine addiction may represent a fundamental tumor survival mechanism with important therapeutic implications.

Glioblastoma multiforme (GBM; World Health Organization astrocytoma grade IV) is the most common primary brain tumor in adults. It represents a highly invasive, proliferative, and aggressive cancer type for which no cure is presently available. Resistance to postoperative radio- and chemotherapy is the main cause of treatment failure in patients with GBM. In patients with newly diagnosed disease, the addition of concomitant and adjuvant temozolomide chemotherapy to postoperative fractionated radiotherapy has recently been shown to increase median survival as well as 2- and 5-year survival rates compared with radiotherapy alone.1,2 During the course of therapy, however, radio- and chemoresistance typically become evident through local tumor recurrence. As a result, only 1 in 4 GBM patients survives 2 years from diagnosis despite aggressive multimodal treatment.1 To improve this poor prognosis, there is a critical need to uncover the molecular basis for the low sensitivity of GBM to chemotherapy. Temozolomide is a prodrug spontaneously converted at physiological pH to its active metabolite 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide, which creates cytotoxic methyl adducts in nitrogen and oxygen atoms in DNA bases.3 However, several independent DNA repair mechanisms, which normally safeguard genome integrity, can facilitate drug resistance and cancer cell survival by removing chemotherapy-induced DNA adducts. For instance, O6-methylguanine adducts are substrates for the ubiquitous DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), whereas the frequent N3-methyladenine and N7-methylguanine adducts are repaired by the base excision repair system in cooperation with poly(ADP-ribose) polymerase–1.4,5 Thus, the anticancer efficacy of temozolomide is highly influenced by cellular DNA repair capacity. Antagonizing specific DNA repair proteins in GBM may prove a promising therapeutic strategy to increase temozolomide cytotoxicity and prolong patient survival. The DNA repair protein human AlkB homolog 2 (ALKBH2) is an oxidative demethylase capable of directly reversing the N1-methyladenine and N3-methylcytosine base lesions in double- and single-stranded DNA.6 Although these lesions are considered weakly mutagenic, they are thought to be highly cytotoxic due to their ability to disturb DNA replication. ALKBH2 is solely expressed in the cell nucleus, where it localizes mainly to replication foci during the S phase of the cell cycle through interaction with proliferating cell nuclear antigen (PCNA) via an ALKBH2 PCNA-interacting motif.6,7 It has therefore been suggested that ALKBH2 may have a role in DNA repair close to the replication fork.8 Embryonal fibroblasts from gene-targeted mice deficient in ALKBH2 have been shown to be incapable of repairing N1-methyladenine in DNA after methyl methanesulfonate treatment.9 As a result, ALKBH2-deficient cells were more sensitive to methyl methansesulfonate exposure than wild type (wt) cells, indicating that ALKBH2 has a functionally important role in the cellular defense against methylating agents. Since N1-methyladenine and N3-methylcytosine are thought to be induced predominantly during the S phase of the cell cycle, the cytotoxic effect of these lesions could be expected to be substantial if left unrepaired in rapid proliferating cells such as cancer cells. However, it is currently unknown to what extent ALKBH2 is expressed in GBM cells and human GBM and whether it can promote resistance to the clinically potent drug temozolomide. In this work, we report that ALKBH2 is highly expressed in GBM cells and human GBM compared with nontumoral human brain (NHB) and that temozolomide exposure may further increase transcription levels of ALKBH2. Moreover, we show that overexpression of ALKBH2 enhances cellular resistance to temozolomide and methyl methanesulfonate but has no effect on cellular sensitivity to nonmethylating agents. We show that silencing of ALKBH2 activity increases cellular sensitivity to both temozolomide and methyl methanesulfonate, suggesting that a high level of ALKBH2 expression could be an important mediator of chemoresistance in human GBM. We also show that nongenotoxic activation of the p53 pathway leads to a substantial downregulation of ALKBH2 at both mRNA and protein levels in GBM cells.

The cancer stem cell hypothesis postulates that tumors arise from, and are maintained by, a small subpopulation of cancer stem cells. This concept has recently become increasingly controversial, following a series of conflicting results. The cell-surface epitope CD133 has been proposed as a brain cancer stem cell marker, whereas a growing number of studies clearly show a tumorigenic potential among CD133− cells as well. Diverging results suggest that assays for isolating cancer stem cells impose a selection bias on which cells are defined as cancer stem cells. Here, we highlight some recent developments, with an emphasis on reports that call for caution in the acceptance of the brain cancer stem cell hypothesis. Cancer Res; 70(21); 8255–8. ©2010 AACR.

Emerging evidence suggests a class of non-coding RNAs termed microRNAs (miRNAs) play a key role in cancer. Since their original discovery in C. elegans in 1993 it has become evident that miRNAs are responsible for an entirely new mechanism of post-transcriptional gene regulation. miRNA expression is widespread in mammalian cells and notably altered in several cancer types. miRNA expression patterns correlate with several aspects of tumorigenesis and miRNA loci have been mapped to frequently altered cancer-associated genomic regions. Inhibition or augmentation of miRNA expression in cancer cells impacts gene expression and affects cell proliferation and survival. Hence, cancer-associated miRNAs may be regarded as a new class of non-coding tumour suppressors and oncogenes capable of regulating several key signalling pathways.

CD133 is a cell surface marker expressed on progenitors of haematopoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor-initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initiated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood-brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter survival. Real-time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy spheroids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregulated, coinciding with the onset of angiogenesis and a shorter survival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved during brain tumor progression.

OBJECTIVE: Glioblastomas (GBM) are highly angiogenic tumors and were initially perceived as good candidates for anti -angiogenic therapy. Bevacuzimab, a monoclonal anti-VEGF antibody, was approved by the FDA in 2009 as monotherapy for recurrent GBM, but recent phase III clinical studies revealed no overall survival benefit, yet with a small improvement on progression free survival. We have recently shown in preclinical GBM models that bevacuzimab treatment increases intratumoral hypoxia with a concomitant up-regulation of HIF 1 alpha. In the present work, we show that the non-psychoactive phytocannabinoid, cannabidiol (CBD) impairs tumor growth in several pre-clinical tumor models. Moreover, we show that CBD down regulates HIF 1 alpha under hypoxic conditions and potentiates the efficacy of anti-angiogenic therapy by inhibiting specific compensatory pathways induced by hypoxia. METHODS: Patient derived GBM spheroids were cultured under hypoxic conditions and treated with CBD. The down regulation of HIF 1 alpha and several of its downstream targets was evaluated. Furthermore GBM spheroids were implanted orthopically in nude rats. The rats were treated with either bevacuzimab, CBD or in combination. Tumor growth was assessed by MR. 18F-FMISO PET was performed to assess tumor hypoxia. RESULTS/CONCLUSIONS: Our results show that CBD treatment down-regulates HIF 1 alpha under hypoxic conditions in vitro and in vivo. Combination treatment with CBD and bevacuzimab decreases tumor growth and intratumoral hypoxia in clinically relevant human GBM xenograft models. Targeting bevacuzimab induced hypoxia by inhibiting HIF 1 alpha might be a promising combinatorial treatment strategy for the treatment of GBM.

2069 Background: Glioblastomas are highly resistant to therapy and carry a poor prognosis. Novel therapeutic strategies are urgently needed to improve patient outcome. This study was designed to id...

Contains fulltext : 50646.pdf (Publisher’s version ) (Closed access) In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.

Metastatic brain tumors are ten times more common than that of all other primary brain tumors combined. Since it is not likely that the metastatic process is based on single genetic events alone, there is a need to find out whether a combination of multiple genes could be responsible for malignant disbursement of cancer cells from the primary site to the brain, in order to develop new treatment strategies. Here, we explored a more global and preventive treatment approach to brain metastases using an established xenograft model. Mice were injected intracardially under ultrasound guidance and followed for eight weeks with whole-body BLI and brain MRI. Tumor-bearing brains, adrenals, ovaries, and femurs were harvested and enzymatically dissociated before sorting out tumor cells by flow cytometry. By using RNA sequencing followed by bioinformatics analysis, we were able to determine a set of 108 genes that were specific for brain metastases, as compared to the metastases from the other organs. We then queried the Connectivity Map database using our 108-gene signature for drugs that could induce an opposite gene expression profile in various cancer cell lines. Three of the top ten drugs, already in use for other clinical conditions, showed significant efficacy in monolayer assays on several metastatic cell lines. We then tested these drugs in vivo, after injecting brain metastatic cell lines intracardially into nod/scid mice. Daily treatment was started immediately, and the animals were followed with whole-body BLI and brain MRI for 10 weeks. The treated animals showed decreased brain metastasis burden, increased survival, and acceptable toxicity profiles. Our results demonstrate a previously unrecognized potential for drug repurposing in the treatment of patients at risk of developing brain metastases or with clinically occult micrometastatic disease. Finding new uses for old drugs can greatly expedite translation to clinical practice.

We have previously reported on a reproducible and predictive animal model that can be used to elucidate the enigmatic biology of melanoma metastasis, a key prerequisite for therapeutic advances. Here, we have used this model to investigate the molecular drivers responsible for metastasis formation in different organs, with a special emphasis on brain-specific features. A human melanoma brain metastasis cell line (H1) was developed in our laboratory, transduced with the genes for green fluorescent protein and luciferase, and injected into the left cardiac ventricle of seven immunodeficient mice. Whole-body bioluminescence imaging (BLI) and brain magnetic resonance imaging was performed weekly to follow the development of metastases. The animals invariably developed both intracranial and extracranial tumors. At about seven weeks, the animals were euthanized and organ involvement was examined in detail by ex vivo BLI. The brain, femurs, adrenals, and ovaries from five random animals were enzymatically dissociated using organ-tailored protocols. Sample yield was validated by fluorescence microscopy prior to fluorescence-activated cell sorting of tumor cells. Three tumor cell samples from the brain, femurs, adrenals, and ovaries of different mice were then analyzed using next-generation RNA-sequencing (NGS) technology. We also performed baseline NGS on the H1 cell line before injection. Taken together, bioinformatics analysis revealed unique molecular signatures of organ-specific metastasis, validating some previously reported metastasis drivers and introducing other genomic features critical to the detrimental momentum of metastatic melanoma. Citation Format: Terje Sundstrøm, Lars Prestegarden, Clifford Tepper, Elizabeth Ingham, Lisa Even, Sarah Johnson, Kai Ove Skaftnesmo, Morten Lund-Johansen, Rolf Bjerkvig, Katherine Ferrara, Frits Thorsen. Organ-specific signatures of melanoma metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2699. doi:10.1158/1538-7445.AM2013-2699

Glioblastoma (GBM) is the most aggressive form of primary brain tumors with a median survival of 15 months. Although angiogenesis is one of the main features of GBMs, non-angiogenic tumor infiltration into brain parenchyma still is the major challenge for therapy. Tumor cells can migrate very far from the main tumor mass and the invasive pattern of tumor subpopulations has not been characterized properly. Epidermal growth factor receptor (EGFR) gene amplification is one of the major mutations of primary GBMs, where multiple copies of the wild-type EGFR gene are present as double minutes. Although studies have proposed a role for EGFR gene amplification in tumor development, the function of EGFR in vivo is not characterized properly mainly due to inefficient tumor models. Here, we report a key role for EGFR wild-type in tumor invasion. In a human GBM xenograft model, we show that tumor cells with EGFR amplification and expression are highly invasive and non-angiogenic. By blocking EGFR activation using Cetuximab and a dominant-negative approach, we show that maintenance of the non-angiogenic, invasive growth pattern is dependent on EGFR function and that downregulation of its activity leads to angiogenic tumor growth. As EGFR amplification and expression is present in 40-60% of GBMs, our results might implicate that activation of EGFR wild-type is one of the major mechanisms of glioblastoma invasion in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-518. doi:1538-7445.AM2012-LB-518

Glioblastoma is the most frequent and most malignant primary brain tumor in adults. Standard first line treatment for glioblastoma patients includes surgery followed by focal fractionated radiotherapy with concomitant and adjuvant administration of the alkylating agent temozolomide. During the course of therapy, however, radio- and chemoresistance typically become evident through local tumour recurrence, and median survival time for glioblastoma patients remains around 15 months. In order to improve this poor prognosis, there is a critical need to recognize the molecular basis for the low sensitivity of glioblastomas towards chemotherapeutic treatment. We have previously reported that elevated expression levels of the DNA repair protein hABH2 may provoke temozolomide resistance in glioma cells. In this work, we explored whether activation of the p53 (encoded by TP53) pathway by the MDM2 antagonist nutlin-3 would influence cellular levels of hABH2 and subsequently chemoresistance. Immunoblotting following nutlin-3 exposure for 24 hours showed increased protein levels of p53, MDM2, p21 and PUMA in glioma cell lines expressing wild type p53, but decreased hABH2 levels in a dose-dependent manner. Real-time quantitative PCR confirmed reduced hABH2 mRNA levels after nutlin-3 therapy, suggesting that hABH2 downregulation occurred at a transcriptional level. Combination therapy with nutlin-3 and temozolomide further enhanced activation of the p53 pathway, diminished levels of hABH2 protein and increased cellular cytotoxicity compared to either agent alone. Our results show that levels of hABH2 can be downregulated by activation of the p53 pathway in glioma cells. This suggests that p53 pathway activation may increase glioma sensitivity to chemotherapy not only by increased transcription of pro-apoptotic genes such as PUMA, but also by reducing levels of DNA repair proteins such as hABH2 that would otherwise promote drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2011-1725

Background and Purpose: Glioblastoma multiforme is the most malignant and frequent primary brain tumour in adults. Current treatment remains insufficient as these tumours display a diffuse infiltrative growth pattern and tend to recur despite extensive debulking surgery followed by radio- and chemotherapy. The methylating agent temozolomide is at present the cornerstone of glioblastoma chemotherapy. However, several independent DNA repair mechanisms can restore the integrity of methylated DNA bases, and thus contribute to temozolomide resistance and subsequent therapy failure. In this work we examined whether human AlkB homologue 2 (hABH2), an oxidative demethylase capable of reversing methyl lesions in DNA, might provoke temozolomide resistance in human glioblastoma cells. Materials and Methods: hABH2 expression was examined in eight established glioma cell lines as well as normal human brain using quantitative real-time PCR. Cells derived from a hABH2 deficient glioblastoma were transfected with a hABH2 plasmid prior to antibiotic selection, and subsequent colonies were screened for hABH2 expression by western blotting. Temozolomide sensitivity was assessed for hABH2 proficient and deficient colonies using a tetrazolium salt assay after 96 hours of drug incubation. Results: mRNA levels of hABH2 were generally 2-4 fold higher in the established cell lines compared to normal human brain. Furthermore, hABH2 overexpressing glioblastoma cells were significantly more resistant to temozolomide than hABH2 deficient cells derived from the same patient. Conclusions: Our results show that hABH2 is expressed at high levels in glioma cells, and that overexpression of this protein mediates temozolomide resistance in vitro. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3641.

Originally known as host defence peptides for their substantial bacteriotoxic effects, many cationic antimicrobial peptides also exhibit a potent cytotoxic activity against cancer cells. Their mode of action is characterized mostly by electrostatic interactions with the plasma membrane, leading to membrane disruption and rapid necrotic cell death. In this work, we have designed a novel cationic peptide of 27 amino acids (Cypep-1), which shows efficacy against a number of cancer cell types, both in vitro and in vivo, while normal human fibroblasts were significantly less affected. Surface plasmon resonance experiments as well as liposome leakage assays monitored by fluorescence spectroscopy revealed a substantial binding affinity of Cypep-1 to negatively charged liposomes and induced significant leakage of liposome content after exposure to the peptide. The observed membranolytic effect of Cypep-1 was confirmed by scanning electron microscopy (SEM) as well as by time-lapse confocal microscopy. Pharmacokinetic profiling of Cypep-1 in rats showed a short plasma half-life after i.v. injection, followed mainly by retention in the liver, spleen and kidneys. Extremely low concentrations within the organs of the central nervous system indicated that Cypep-1 did not pass the blood-brain-barrier. Local treatment of 4T1 murine mammary carcinoma allografts by means of a single local bolus injection of Cypep-1 led to a significant reduction of tumour growth in the following weeks and prolonged survival. Detailed histological analysis of the treated tumours revealed large areas of necrosis. In sum, our findings show that the novel cationic peptide Cypep-1 displays a strong cytolytic activity against cancer cells both in vitro and in vivo and thus holds a substantial therapeutic potential.

Glioblastoma multiforme (GBM) is highly resistant to therapy and carries a dismal prognosis. Standard of care includes surgery followed by radiotherapy (RT) with concurrent and adjuvant chemotherapy with temozolomide (TMZ). The addition of TMZ to RT increases the overall survival by approximately 3 months compared to RT alone, but there are virtually no long-term survivors. In this work we sought to identify small-molecule sensitizers to TMZ in GBM by integrating a genome-wide lentiviral shRNA library screen with the Connectivity Map database. We first transfected the human GBM cell line U87 with a pooled RNAi library targeting approximately 5000 genes, and incubated the cells at three sub-lethal TMZ concentrations or in control medium. DNA was extracted from both treated and untreated cells at 12 days. Barcodes identifying each shRNA were amplified by PCR and quantified using next-generation sequencing. Individual silencing of 292 genes lead to significantly reduced cell growth in the treated population while silencing of 414 genes increased cellular viability compared to the control group. The Connectivity Map database was then interrogated using these results to identify small-molecule inhibitors that would induce analogous changes in gene expression. This integrated high-throughput approach identified a broad range of compounds with potential cytotoxic activity such as the anti-psychotic thioridazine, the phytoestrogen resveratrol, the antibiotic furazolidone, and the proteasomal inhibitor MG-262. Upon validation in low-throughput cytotoxicity assays, we found these compounds effective as single-agent therapies, but also synergistic with concomitant TMZ therapy. Several of these compounds have previously been implicated in anti-cancer therapy, but not as synthetic lethal partners of chemotherapeutic drugs. Interestingly, we found a prominent cytotoxic effect of the dopamine receptor antagonist thioridazine when given as monotherapy and as combination therapy with TMZ. The therapeutic potential of thioridazine and concomitant TMZ in vivo is currently being explored in a clinically relevant intracranial GBM animal model.